I. Field of the Invention
The present invention relates generally to methods, compositions and kits for making unidirectionally or bidirectionally truncated polypeptides. The invention has broad applicability for research, industrial, and therapeutic uses, such as, but not limited to uses for engineering of proteins, including enzymes, identification of protein domains, changing protein functionality, epitope mapping, and for preparation of antigenic peptides for production of vaccines.
II. Description of Related Art
The introduction of deletions in nucleic acid sequences is a widely used method in molecular biology to study polypeptides encoded by the nucleic acid sequences.
Multiple procedures have been developed to generate deletions in nucleic acids, including procedures disclosed by Dunn et al. (U.S. Pat. No. 5,928,908; U.S. Pat. No. 5,968,768; and U.S. Pat. No. 6,248,569); Shen et al. (U.S. Pat. No. 5,356,773); Yohda et al. (DNA Research, 2: 175-181, 1995); Zhu and Marshall (BioTechniques, 18: 222-224, 1995); and Henikoff et al. (Gene, 28: 351-359, 1984 and U.S. Pat. No. 4,843,003). Other procedures for generating deletions have utilized variations of PCR (e.g., Pues et al., Nucleic Acids Res. 25: 1303-1304, 1997). All of the above are incorporated herein by reference in their entireties.
The current techniques for generating deletions in a target sequence are time-consuming and wasteful and what is needed are procedures to make deletions of nucleic acids that encode proteins that are less time-consuming and more efficient. What is further needed are procedures that allow the generation of deletions without the need to first clone the nucleic acid sequence of interest into a vector. Also, what is needed are methods to make large quantities of deletions in a random fashion, so that surprising and unexpected results can be obtained.
DNA transposons are mobile elements that can move from one position in a genome to another. In nature, transposons play roles in evolution as a result of their movements within and between genomes. Transposons are relatively simple genetic systems, consisting of some genetic sequence bounded by inverted terminal repeats and a transposase enzyme that acts to cut the transposon out of one source of DNA and paste it into another DNA sequence. Autonomous transposons carry the transposase gene inside the transposon whereas non-autonomous transposons require another source of transposase for their mobilization.
Multiple studies of transposition have been have been published (Devine et al., U.S. Pat. No. 5,677,170; Devine et al., U.S. Pat. Nos. 5,728,551 and 5,968,785; Hackett et al., International Patent Application No. WO 98/40510; Plasternak et al., International Patent Application No. WO 97/29202; Reznikoff et al., International Patent Application No. WO 98/10077; Craig, International Patent Application No. WO 98/37205; Strathman et al., Proc. Nat. Acad. Sci. USA 88: 1247-1250, 1991; Phadnis et al., Proc. Nat. Acad. Sci. USA 86: 5908-5912, 1989; Way et al., Gene 32: 269-279, 1984; Kleckner et al., Method. Enzymol. 204: 139-180, 1991; Lee et al., Proc. Nat. Acad. Sci. USA 84: 7876, 1987; Brown et al., Cell, 49: 347-356, 1987; Eichinger et al., Cell, 54: 955-966, 1988; Eichinger et al., Genes Dev., 4: 324-330, 1990; all of which are incorporated herein by reference in their entireties.
A number of studies have reported on deletions generated by transposons or the use of transposons to generate deletions for using in nucleic acid sequencing. Among these are articles by Ahmed (J. Mol. Biol., 178: 941-948, 1984); Hattori et al. (Nucleic Acids Res. 25: 1802-1808, 1997); Jilk et al. (J. Bacteriology, 175: 1264-1271, 1993); Krishnan et al. (Nucleic Acids Res. 23: 117-122, 1995); Morita et al., (DNA Research, 3: 431-433, 1996); Sugino et al. (U.S. Pat. No. 6,265,159 BI); Wang et al. (J. Bacteriology, 176: 6348-6354, 1990); and Wang et al. (Proc. Natl. Acad. Sci., 90: 7874-7878, 1993), all of which are incorporated herein by reference in their entireties.
Methods for using vectors with specially positioned and oriented transposon end sequences and a transposase were used for making unidirectional deletions in a target sequence cloned into the vector for a variety of purposes, as described by York et al. (Nucleic Acids Res. 26: 1927, 1998 and U.S. Pat. No. 5,948,622). These researchers also described how the methods could be used for making unidirectionally truncated polypeptides.
In U.S. Pat. No. 6,593,113, which is incorporated herein by reference in its entirety, Tenkanen et al. describe the use of a transposition reaction and a subsequent amplification reaction for providing templates for DNA sequencing. The inventors do not envision a method for making truncated polypeptides.
WO03/87370, incorporated herein by reference in its entirety, teaches a method for producing deletion derivatives of polypeptides by inserting transposons that contain translation stop signals into target nucleic acid sequences that have been cloned into vectors. The need to first clone a nucleic acid sequence of interest is time consuming and lacks efficiency.